Cyanophenoxy carboxylic acid compounds and compositions for delivering active agents

ABSTRACT

Cyanophenoxy carboxylic acid compounds and compositions for the delivery of active agents are provided. Methods of administration, treatment of disease and preparation are provided as well.

This application is a national phase of PCT Application No.PCT/US01/41984, filed Sep. 5, 2001, which was published in English asInternational Publication No. WO 02/20466 and claims the benefit of U.S.Provisional Application Nos. 60/230,331 and 60/237,235, filed Sep. 6,2000 and Oct. 2, 2000, respectively, both of which are hereinincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to cyanophenoxy carboxylic acid compoundsfor delivering active agents, such as biologically or chemically activeagents, to a target. These compounds are well suited for formingnon-covalent mixtures with active agents for oral, intracolonic,pulmonary, and other routes of administration to animals. Methods forthe preparation and administration of such compositions are alsodisclosed.

BACKGROUND OF THE INVENTION

Conventional means for delivering active agents are often severelylimited by biological, chemical, and physical barriers. Typically, thesebarriers are imposed by the environment through which delivery occurs,the environment of the target for delivery, and/or the target itself.Biologically and chemically active agents are particularly vulnerable tosuch barriers.

In the delivery to animals of biologically active and chemically activepharmacological and therapeutic agents, barriers are imposed by thebody. Examples of physical barriers are the skin, lipid bi-layers andvarious organ membranes that are relatively impermeable to certainactive agents but must be traversed before reaching a target, such asthe circulatory system. Chemical barriers include, but are not limitedto, pH variations in the gastrointestinal (GI) tract and degradingenzymes.

These barriers are of particular significance in the design of oraldelivery systems. Oral delivery of many biologically or chemicallyactive agents would be the route of choice for administration to animalsif not for biological, chemical, and physical barriers. Among thenumerous agents which are not typically amenable to oral administrationare biologically or chemically active peptides, such as calcitonin andinsulin; polysaccharides, and in particular mucopolysaccharidesincluding, but not limited to, heparin; heparinoids; antibiotics; andother organic substances. These agents may be rapidly renderedineffective or destroyed in the gastro-intestinal tract by acidhydrolysis, enzymes, and the like. In addition, the size and structureof macromolecular drugs may prohibit absorption.

Earlier methods for orally administering vulnerable pharmacologicalagents have relied on the co-administration of adjuvants (e.g.,resorcinols and non-ionic surfactants such as polyoxyethylene oleylether and n-hexadecylpolyethylene ether) to increase artificially thepermeability of the intestinal walls, as well as the co-administrationof enzymatic inhibitors (e.g., pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymaticdegradation. Liposomes have also been described as drug delivery systemsfor insulin and heparin. However, broad spectrum use of such drugdelivery systems is precluded because: (1) the systems require toxicamounts of adjuvants or inhibitors; (2) suitable low molecular weightcargos, i.e., active agents, are not available; (3) the systems exhibitpoor stability and inadequate shelf life; (4) the systems are difficultto manufacture; (5) the systems fail to protect the active agent(cargo); (6) the systems adversely alter the active agent; or (7) thesystems fail to allow or promote absorption of the active agent.

Proteinoid microspheres have been used to deliver pharmaceuticals. See,for example, U.S. Pat. Nos. 5,401,516; 5,443,841; and Re. 35,862. Inaddition, certain modified amino acids have been used to deliverpharmaceuticals. See, for example, U.S. Pat. Nos. 5,629,020; 5,643,957;5,766,633; 5,776,888; and 5,866,536.

More recently, a polymer has been conjugated to a modified amino acid ora derivative thereof via a linkage group to provide for polymericdelivery agents. The modified polymer may be any polymer, but preferredpolymers include, but are not limited to, polyethylene glycol (PEG), andderivatives thereof. See, for example, International Patent PublicationNo. WO 00/40203.

However, there is still a need for simple, inexpensive delivery systemswhich are easily prepared and which can deliver a broad range of activeagents by various routes.

SUMMARY OF THE INVENTION

The present invention provides compounds and compositions whichfacilitate the delivery of active agents. Delivery agent compounds ofthe present invention include those having the following formula:

and salts thereofwherein

R¹, R², R³, R⁴ and R⁵ are independently H, —CN, —OH, —OCH₃, or halogen,at least one of R¹, R², R³, R⁴ and R⁵ being —CN; and

R⁶ is C₁–C₁₂ linear or branched alkylene, alkenylene, arylene,alkyl(arylene) or aryl(alkylene),

with the proviso that when R¹ is —CN, R⁴ is H or —CN, and R², R³, and R⁵are H, then R⁶ is not methylene ((CH₂)₁).

In a preferred embodiment, R¹ is H or —CN. In another preferredembodiment, R⁴ is H, —CN, or a halogen. In another preferred embodiment,the halogen is Cl.

Preferably, R⁶ is C₁–C₉ alkylene. More preferably R is C₂–C₉ alkylene.According to a more preferred embodiment, R⁶ is C₄–C₇ alkylene.According to another preferred embodiment, R⁶ is (CH₂)₁, (CH₂)₃, (CH₂)₄,(CH₂)₅, (CH₂)₇, or (CH₂)₉.

In a preferred embodiment, R¹ is —CN. Preferably, R², R³, R⁴ and R⁵ areH or halogen, preferably Cl. Preferably, R⁶ is (CH₂)_(n) where n is1–12, preferably 2–9, more preferably 3–7, and more preferably 7 or R⁶is —(CH₂)-para-phenylene.

In another preferred embodiment, R³ is —CN. Preferably, R¹, R², R⁴ andR⁵ are H or halogen, preferably Cl. Preferably, R⁶ is (CH₂)_(n) and n is1–12, preferably 2–9, more preferably 3–7, and more preferably 7.

In another preferred embodiment, the compound comprises the compounds ofTable 1 or salts thereof or mixtures thereof:

TABLE 1 Delivery Agent Compounds Cpd # R¹ R² R³ R⁴ R⁵ R⁶ 1 CN H H H H(CH₂)₁ 2 CN H H H H (CH₂)₃ 3 CN H H H H (CH₂)₄ 4 CN H H H H (CH₂)₅ 5 CNH H H H (CH₂)₇ 6 CN H H H H (CH₂)₉ 7 CN H Cl H H (CH₂)₄ 8 H H CN H H(CH₂)₇ 9 CN H H H H (CH₂)₁-para-phenyl-

The chemical structures of compounds 1–9 are shown below:

and salts thereof or mixture thereof.

The invention also provides a composition comprising at least one of thedelivery agent compounds of the formulas above, and at least one activeagent. These compositions deliver active agents to selected biologicalsystems in increased or improved bioavailability of the active agentcompared to administration of the active agent without the deliveryagent compound.

Also provided are dosage unit forms comprising the compositions. Thedosage unit may be in the form of a liquid or a solid, such as a tablet,capsule or particle, including a powder or sachet.

Another embodiment is a method for administering an active agent to ananimal in need of the active agent, by administering a compositioncomprising at one of the delivery agent compounds of the formulae aboveand the active agent to the animal. Preferred routes of administrationinclude the oral, intracolonic and pulmonary routes.

Yet another embodiment is a method of treating a disease or forachieving a desired physiological effect in an animal in need thereof byadministering an effective amount of the composition of the presentinvention.

Yet another embodiment is a method of preparing a composition of thepresent invention by mixing at least one delivery agent compound of theformulae above, and at least one active agent.

DETAILED DESCRIPTION OF THE INVENTION

Delivery Agent Compounds

The terms “alkyl” and “alkenyl” as used herein include linear andbranched alkyl and alkenyl substituents, respectively.

The delivery agent compounds may be in the form of the carboxylic acidor salts thereof. Suitable salts include, but are not limited to,organic and inorganic salts, for example alkali-metal salts, such assodium, potassium and lithium; alkaline-earth metal salts, such asmagnesium, calcium or barium; ammonium salts; basic amino acids, such aslysine or arginine; and organic amines, such as dimethylamine orpyridine. Preferably, the salts are sodium salts. The salts may be mono-or multi-valent salts, such as monosodium salts and di-sodium salts. Thesalts may also be solvates, including ethanol solvates, and hydrates.

Salts of the delivery agent compounds of the present invention may beprepared by methods known in the art. For example, sodium salts may beprepared by dissolving the delivery agent compound in ethanol and addingaqueous sodium hydroxide.

In addition, poly amino acids and peptides comprising one or more ofthese delivery agent compounds may be used.

An amino acid is any carboxylic acid having at least one free aminegroup and includes naturally occurring and synthetic amino acids. Polyamino acids are either peptides (which are two or more amino acidsjoined by a peptide bond) or are two or more amino acids linked by abond formed by other groups which can be linked by, e.g., an ester or ananhydride linkage. Peptides can vary in length from dipeptides with twoamino acids to polypeptides with several hundred amino acids. One ormore of the amino acids or peptide units may be acylated or sulfonated.

The compounds described herein may be derived from amino acids and canbe readily prepared from amino acids by methods within the skill ofthose in the art based upon the present disclosure and the methodsdescribed in International Patent Publication Nos. WO 96/30036 and WO97/36480 and U.S. Pat. Nos. 5,643,957 and 5,650,386. For example, thedelivery agent compounds may be prepared by reacting the single aminoacid with the appropriate acylating or amine-modifying agent, whichreacts with a free amino moiety present in the amino acid to formamides. Protecting groups may be used to avoid unwanted side reactionsas would be known to those skilled in the art. With regard to protectinggroups, reference is made to T. W. Greene, Protecting Groups in OrganicSynthesis, Wiley, New York (1981), the disclosure of which is herebyincorporated herein by reference.

The delivery agent compound may be purified by recrystallization or byfractionation on one or more solid chromatographic supports, alone orlinked in tandem. Suitable recrystallization solvent systems include,but are not limited to, ethanol, water, heptane, ethyl acetate,acetonitrile, methanol, tetrahydrofuran and mixtures thereof.Fractionation may be performed on a suitable chromatographic supportsuch as alumina, using methanol/n-propanol mixtures as the mobile phase;reverse phase chromatography using trifluoroacetic acid/acetonitrilemixtures as the mobile phase; and ion exchange chromatography usingwater or an appropriate buffer as the mobile phase. When anion exchangechromatography is performed, preferably a 0–500 mM sodium chloridegradient is employed.

The delivery agent compound may contain a polymer conjugated to it by alinkage group selected from the group consisting of —NHC(O)NH—,—C(O)NH—, —NHC(O), —OOC—, —COO—, —NHC(O)O—, —OC(O)NH—, —CH₂NH —NHCH₂—,—CH₂NHC(O)O—, —OC(O)NHCH₂—, —CH₂NHCOCH₂O—, —OCH₂C(O)NHCH₂—,—NHC(O)CH₂O—, —OCH₂C(O)NH—, —NH—, —O—, and carbon-carbon bond. Accordingto one preferred embodiment, the polymeric delivery agent is not apolypeptide or polyamino acid. The polymer may be any polymer including,but not limited to, alternating copolymers, block copolymers and randomcopolymers, which are safe for use in mammals. Preferred polymersinclude, but are not limited to, polyethylene; polyacrylates;polymethacrylates; poly(oxyethylene); poly(propylene); polypropyleneglycol; polyethylene glycol (PEG); and derivatives thereof andcombinations thereof. The molecular weight of the polymer typicallyranges from about 100 to about 200,000 daltons. The molecular weight ofthe polymer preferably ranges from about 200 to about 10,000 daltons. Inone embodiment, the molecular weight of the polymer ranges from about200 to about 600 daltons and more preferably ranges from about 300 toabout 550 daltons.

Active Agents

Active agents suitable for use in the present invention includebiologically active agents and chemically active agents, including, butnot limited to, pesticides, pharmacological agents, and therapeuticagents. Suitable active agents include those that are rendered lesseffective, ineffective or are destroyed in the gastrointestinal tract byacid hydrolysis, enzymes and the like. Also included as suitable activeagents are those macromolecular agents whose physiochemicalcharacteristics, such as, size, structure or charge, prohibit or impedeabsorption when dosed orally.

For example, biologically or chemically active agents suitable for usein the present invention include, but are not limited to, proteins;polypeptides; peptides; hormones; polysaccharides, and particularlymixtures of muco-polysaccharides; carbohydrates; lipids; small polarorganic molecules (i.e. polar organic molecules having a molecularweight of 500 daltons or less); other organic compounds; andparticularly compounds which by themselves do not pass (or which passonly a fraction of the administered dose) through the gastro-intestinalmucosa and/or are susceptible to chemical cleavage by acids and enzymesin the gastro-intestinal tract; or any combination thereof.

Further examples include, but are not limited to, the following,including synthetic, natural or recombinant sources thereof: growthhormones, including human growth hormones (hGH), recombinant humangrowth hormones (rhGH), bovine growth hormones, and porcine growthhormones; growth hormone releasing hormones; growth hormone releasingfactor, interferons, including α, β and γ; interleukin-1; interleukin-2;insulin, including porcine, bovine, human, and human recombinant,optionally having counter ions including zinc, sodium, calcium andammonium; insulin-like growth factor, including IGF-1; heparin,including unfractionated heparin, heparinoids, dermatans, chondroitins,low molecular weight heparin, very low molecular weight heparin andultra low molecular weight heparin; calcitonin, including salmon, eel,porcine and human; erythropoietin; atrial naturetic factor; antigens;monoclonal antibodies; somatostatin; protease inhibitors;adrenocorticotropin, gonadotropin releasing hormone; oxytocin;leutinizing-hormone-releasing-hormone; follicle stimulating hormone;glucocerebrosidase; thrombopoietin; filgrastim; prostaglandins;cyclosporin; vasopressin; cromolyn sodium (sodium or disodiumchromoglycate); vancomycin; desferrioxamine (DFO); bisphosphonates,including alendronate, tiludronate, etidronate, clodronate, pamidronate,olpadronate, and incadronate; parathyroid hormone (PTH), including itsfragments; antimicrobials, including antibiotics, anti-bacterials andanti-fungal agents; vitamins; analogs, fragments, mimetics orpolyethylene glycol (PEG)-modified derivatives of these compounds; orany combination thereof. Non-limiting examples of antibiotics includegram-positive acting, bacteriocidal, lipopeptidal and cyclic peptidalantibiotics, such as daptomycin and analogs thereof.

Delivery Systems

The composition of the present invention comprises one or more deliveryagent compounds of the present invention (including their salts andpolymeric derivatives), and one or more active agents. In oneembodiment, one or more of the delivery agent compounds, or salts ofthese compounds, or poly amino acids or peptides of which thesecompounds or salts form one or more of the units thereof, may be used asa delivery agent by mixing with the active agent prior to administrationto form an administration composition.

The administration compositions may be in the form of a liquid. Thesolution medium may be water (for example, for salmon calcitonin,parathyroid hormone, and erythropoietin), 25% aqueous propylene glycol(for example, for heparin) and phosphate buffer (for example, for rhGH).Other dosing vehicles include polyethylene glycol. Dosing solutions maybe prepared by mixing a solution of the delivery agent compound with asolution of the active agent, just prior to administration. Alternately,a solution of the delivery agent compound (or active agent) may be mixedwith the solid form of the active agent (or delivery agent compound).The delivery agent compound and the active agent may also be mixed asdry powders. The delivery agent compound and the active agent can alsobe admixed during the manufacturing process.

The dosing solutions may optionally contain additives such as phosphatebuffer salts, citric acid, glycols, or other dispersing agents.Stabilizing additives may be incorporated into the solution, preferablyat a concentration ranging between about 0.1 and 20% (w/v).

The administration compositions may alternately be in the form of asolid, such as a tablet, capsule or particle, such as a powder orsachet. Solid dosage forms may be prepared by mixing the solid form ofthe delivery agent compound with the solid form of the active agent.Alternately, a solid may be obtained from a solution of the deliveryagent compound and active agent by methods known in the art, such asfreeze-drying (lyophilization), precipitation, crystallization and soliddispersion.

The administration compositions of the present invention may alsoinclude one or more enzyme inhibitors. Such enzyme inhibitors include,but are not limited to, compounds such as actinonin or epiactinonin andderivatives thereof. Other enzyme inhibitors include, but are notlimited to, aprotinin (Trasylol) and Bowman-Birk inhibitor.

The amount of active agent used in an administration composition of thepresent invention is an amount effective to accomplish the purpose ofthe particular active agent for the target indication. The amount ofactive agent in the compositions typically is a pharmacologically,biologically, therapeutically, or chemically effective amount. However,the amount can be less than that amount when the composition is used ina dosage unit form because the dosage unit form may contain a pluralityof delivery agent compound/active agent compositions or may contain adivided pharmacologically, biologically, therapeutically, or chemicallyeffective amount. The total effective amount can then be administered incumulative units containing, in total, an effective amount of the activeagent.

The total amount of active agent to be used can be determined by methodsknown to those skilled in the art. However, because the compositions ofthe invention may deliver active agents more efficiently thancompositions containing the active agent alone, lower amounts ofbiologically or chemically active agents than those used in prior dosageunit forms or delivery systems can be administered to the subject, whilestill achieving the same blood levels and/or therapeutic effects.

The presently disclosed delivery agent compounds facilitate the deliveryof biologically and chemically active agents, particularly in oral,intranasal, sublingual, intraduodenal, subcutaneous, buccal,intracolonic, rectal, vaginal, mucosal, pulmonary, transdermal,intradermal, parenteral, intravenous, intramuscular and ocular systems,as well as traversing the blood-brain barrier.

Dosage unit forms can also include any one or combination of excipients,diluents, disintegrants, lubricants, plasticizers, colorants,flavorants, taste-masking agents, sugars, sweeteners, salts, and dosingvehicles, including, but not limited to, water, 1,2-propane diol,ethanol, olive oil, or any combination thereof.

The compounds and compositions of the subject invention are useful foradministering biologically or chemically active agents to any animals,including but not limited to, birds such as chickens; mammals, such asrodents, cows, pigs, dogs, cats, primates, and particularly humans; andinsects.

The system is particularly advantageous for delivering chemically orbiologically active agents that would otherwise be destroyed or renderedless effective by conditions encountered before the active agent reachesits target zone (i.e. the area in which the active agent of the deliverycomposition is to be released) and within the body of the animal towhich they are administered. Particularly, the delivery agent compoundsand compositions of the present invention are useful in orallyadministering active agents, especially those that are not ordinarilyorally deliverable, or those for which improved delivery is desired.

The compositions comprising the delivery agent compounds and activeagents have utility in the delivery of active agents to selectedbiological systems and in an increased or improved bioavailability ofthe active agent compared to administration of the active agent withoutthe delivery agent. Delivery can be improved by delivering more activeagent over a period of time, or in delivering active agent in aparticular time period (such as to effect quicker or delayed delivery),or in delivering the active agent at a specific time, or over a periodof time (such as sustained delivery).

Another embodiment of the present invention is a method for thetreatment or prevention of a disease or for achieving a desiredphysiological effect, such as those listed in the table below, in ananimal by administering the composition of the present invention.Specific indications for active agents can be found in the Physicians'Desk Reference (54^(th) Ed., 2000, Medical Economics Company, Inc.,Montvale, N.J.), which is herein incorporated by reference. The activeagents in the table below include their analogs, fragments, mimetics,and polyethylene glycol-modified derivatives.

Disease and Physiological Active Agent Effect Growth hormones Growthdisorders Interferons, including α, β and Viral infection, including γ.chronic cancer and multiple sclerosis Interleukin-1; interleukin-2.Viral infection; cancer Insulin; Insulin-like growth Diabetes factorIGF-1. Heparin Thrombosis; prevention of blood coagulation Calcitonin.Osteoporosis; diseases of the bone Erythropoietin Anemia Atrialnaturetic factor Vasodilation Antigens Infection Monoclonal antibodiesTo prevent graft rejection; cancer Somatostatin Bleeding ulcer; erosivegastritis Protease inhibitors AIDS Adrenocorticotropin High cholesterol(to lower cholesterol) Gonadotropin releasing hormone Ovulatorydisfunction (to stimulate ovulation) Oxytocin Labor disfunction (tostimulate contractions) Leutinizing-hormone-releasing- Regulatereproductive function hormone; follicle stimulating hormoneGlucocerebrosidase Gaucher disease (to metabolize lipoprotein)Thrombopoietin Thrombocytopenia Filgrastim Reduce infection inchemotherapy patients Prostaglandins Hypertension Cyclosporin Transplantrejection Vasopressin Bed-wetting; antidiuretic Cromolyn sodium;Vancomycin Asthma; allergies Desferrioxamine (DFO) Iron overloadParathyroid hormone (PTH), Osteoporosis; including its fragments.Diseases of the bone Antimicrobials Infection including gram- positivebacterial infection Vitamins Vitamin deficiencies BisphosphonatesOsteoporosis; Paget's disease; Inhibits osteoclasts

For example, one embodiment of the present invention is a method fortreating a patient suffering from or susceptible to diabetes byadministering insulin and at least one of the delivery agent compoundsof the present invention.

Following administration, the active agent present in the composition ordosage unit form is taken up into the circulation. The bioavailabilityof the agent is readily assessed by measuring a known pharmacologicalactivity in blood, e.g. an increase in blood clotting time caused byheparin, or a decrease in circulating calcium levels caused bycalcitonin. Alternately, the circulating levels of the active agentitself can be measured directly.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples illustrate the invention without limitation. Allparts are given by weight unless otherwise indicated.

Proton nuclear magnetic resonance (¹H NMR) analyses for the compoundslisted below were conducted on a 300 MHz Bruker spectrometer usingdimethyl sulfoxide (DMSO-d₆) as the solvent unless otherwise indicated.

EXAMPLE 1 Compound Preparation

1a: Preparation of Compound 1

A 3-neck 300 mL round-bottomed flask equipped with a reflux condenser,magnetic stir bar and a nitrogen inlet was charged with 5 g (1 equiv.)of 2-hydroxybenzonitrile, absolute ethanol 150 mL, and 15.7 mL (1equivalent) of sodium ethoxide. This mixture was stirred at 25° C. for15 minutes. Ethyl bromoacetate (4.6 mL, 1 equivalent) was then addeddropwise over 10 minutes. The resulting mixture was heated to reflux(75° C.) for 72 hours.

The reaction mixture was cooled and the solids filtered off. The solventwas removed on a rotary evaporator. The crude residue was dissolved inmethylene chloride (250 mL) and washed with saturated NaHCO₃ (3×100mL)IH₂O (1×100 mL) and brine (1×50 mL). The organic layer was dried togive the crude ester. The crude material was then dissolved in ethanol(150 mL) and water (10 mL). LiOH (4 g) was added and the resultingmixture was heated to reflux (75° C.) for 3 hours. The solution wascooled and the solvent removed. 100 mL of H₂O was added and the aqueoussolution was acidified to a pH of about 2 with concentrated hydrochloricacid. The solution was cooled in a 4° C. refrigerator. A tan coloredsolid precipitated. This material was collected by vacuum filtration anddried on the high vacuum overnight to give 6.71 g of the product,3-(2-cyanophenoxy)acetic acid (90% yield). Melting point: 179–181° C.Molecular Formula: C₉H₇NO₃. Combustion analysis: % C: 61.02 (calc'd),60.69 (found); % H: 3.98(calc'd), 3.98 (found); % N: 7.91 (calc'd), 7.66(found).

1b. Preparation of Compound 2

A 3-neck 300 mL round-bottomed flask equipped with a reflux condenser,magnetic stir bar and an N₂ inlet was charged with 5 g (1 equiv.) of2-hydroxybenzonitrile, absolute ethanol 150 mL, and 15.7 mL (1equivalent) of sodium ethoxide. This mixture was stirred at 25° C. for15 minutes. Ethyl 4-bromobutyrate (6.0 mL, 1 equivalent) was then addeddropwise over 10 minutes. The resulting mixture was heated to reflux(75° C.) for 72 hours.

The reaction mixture was cooled and the solids filtered off. The solventwas removed on a rotary evaporator. The crude residue was dissolved inmethylene chloride (300 mL) and washed with saturated NaHCO₃ (2×100 mL),H₂O (1×100 mL) and brine (1×50 mL). The organic layer was dried to givethe crude ester. The crude material was then dissolved in ethanol (150mL) and water (10 mL). LiOH (5 grams) was added and the resultingmixture was heated to reflux (75° C.) for 3 hours. The solution wascooled and the solvent removed. 75 mL of H₂O was added and the aqueoussolution was acidified to a pH of about 2 with concentrated HCl. Thesolution was cooled in a 4° C. refrigerator. A tan colored solidprecipitated. This material was collected by vacuum filtration and driedon the high vacuum overnight to give the product,4-(2-cyanophenoxy)butanoic acid (71% yield). Melting point: 127–128° C.Molecular Formula: C₁₁H₁₁NO₃. Combustion analysis: % C: 64.38 (calc'd),64.01 (found); % H: 5.4(calc'd), 5.2(found); % N: 6.83 (calc'd), 6.74(found).

1c: Preparation of Compound 3

A 3-neck 300 mL round-bottomed flask equipped with a reflux condenser,magnetic stir bar and a nitrogen inlet was charged with 4 g (1equivalent) of 2-hydroxybenzonitrile, absolute ethanol 150 mL, and 12.5mL (1 equivalent) of sodium ethoxide. This mixture was stirred at 25° C.for 15 minutes. Ethyl 5-bromovalerate (5.3 mL, 1 equivalent) was thenadded dropwise over 10 minutes. The resulting mixture was heated toreflux (80° C.) for 72 hours. The reaction mixture was cooled and thesolids filtered off. The solvent was removed on a rotary evaporator. Thecrude residue was dissolved in methylene chloride (200 mL) and washedwith saturated NaHCO₃ (2×100 mL), H₂O (1×50 mL) and brine (1×50 mL). Theorganic layer was dried to give the crude ester. The crude material wasthen dissolved in ethanol (150 mL) and water (10 mL). LiOH (3.5 g) wasadded and the resulting mixture was heated to reflux (80° C.) for 3hours. The solution was cooled and the solvent removed. 75 mL of H₂O wasadded and the aqueous solution was acidified to a pH of approximately 2with concentrated HCl. The flask was cooled by placing it in a 4° C.refrigerator for 4 hours. A tan colored solid precipitated. Thismaterial was collected by vacuum filtration and dried on the high vacuumovernight to give 6.2 g of material (84% yield). This material wasfurther purified by recrystallization from ethyl acetate/hexanes(approximately 95/5) to give 5.3 g of 5-(2-cyanophenoxy)pentanoic acid.Melting point: 87–89° C. Combustion analysis: % C: 65.74 (calc'd), 65.52(found); % H: 5.98 (calc'd), 5.86 (found); % N: 6.39 (calc'd), 6.38(found). ¹H NMR Analysis: (d₆-DMSO): δ 12.0, s, 1H (COOH); δ 7.73–7.62,m, 2H (aromatic CH's ortho and para to CN); δ 7.25, d, 1H, J=8.5 Hz(aromatic CH para to OR); δ 7.10, dt, 1H, J=0.7 and 6.8 Hz (aromatic CHortho to OR); δ 4.16, t, 2H, J=7.5 Hz (CH₂ α to O); δ 2.33, t, 2H, J=7.2Hz (CH₂ α to COOH); δ 1.80–1.64, m, 4H (remaining aliphatic CH₂'s).

1d: Preparation of Compound 4

A 3-neck 300 mL round-bottomed flask equipped with a reflux condenser,magnetic stir bar and an N₂ inlet was charged with 5 g (1 equivalent) of2-hydroxybenzonitrile, absolute ethanol 150 mL, and 15.7 mL (1equivalent) of sodium ethoxide. This mixture was stirred at 25° C. for15 minutes. Ethyl 6-bromohexanoate (7.5 mL, 1 equiv.) was then addeddropwise over 10 minutes. The resulting mixture was heated to reflux(75° C.) for 72 hours.

The reaction mixture was cooled and the solids filtered off. The solventwas removed on a rotary evaporator. The crude residue was dissolved inmethylene chloride (300 mL) and washed with saturated NaHCO₃ (3×100 mL),H₂O (1×100 mL) and brine (1×100 mL). The organic layer was dried to givethe crude ester. The crude material was then dissolved in ethanol (150mL) and water (15 mL). LiOH (7 g) was added and the resulting mixturewas heated to reflux (75° C.) for 2 hours. The solution was cooled andthe solvent removed. 125 mL of H₂O was added and the aqueous solutionwas acidified to pH˜2 with concentrated HCl. The solution was cooled ina 4° C. refrigerator. A tan colored solid precipitated. This materialwas collected by vacuum filtration and dried on the high vacuumovernight to give the crude acid. This material was further purified byrecrystallization from ethyl acetate/hexanes (95/5) to give 6.81 g of6-(2-cyanophenoxy)hexanoic acid (70% yield). Melting point: 77–80° C.Karl Fisher: 1.26% H₂O. Molecular Formula with H₂O: C13H15NO3*0.1652.Combustion analysis: % C: 66.09 (calc'd), 66.19 (found); % H:6.54(calc'd), 6.36 (found); % N: 5.93 (calc'd), 5.9 (found). ¹H NMRAnalysis: (d₆-DMSO): δ 12.0, s, 1H; 7.72–7.61, m, 2H; 7.25, d, 1H; 7.10,dt, 1H; 4.14, t, 2H; 2.26, t, 2H; 1.80–1.41, m, 6H.

1e. Preparation of Compound 5

A 3-neck 300 mL round-bottomed flask equipped with a reflux condenser,magnetic stir bar and an N₂ inlet was charged with 10 g (1 equivalent)of 2-hydroxybenzonitrile, absolute ethanol 400 mL, and 31.3 mL (1equivalent) of sodium ethoxide. This mixture was stirred at 25° C. for15 minutes. Ethyl 8-bromooctanoate (21 g, 1 equivalent) was then addeddropwise over 15 minutes. The resulting mixture was heated to reflux(80° C.) for 72 hours.

The reaction mixture was cooled and the solids filtered off. The solventwas removed on a rotary evaporator. The crude residue was dissolved inmethylene chloride (400 mL) and washed with saturated NaHCO₃ (3×100 mL),bleach (1×100 mL), H₂O (1×50 mL) and brine (1×50 mL). The organic layerwas dried to give the crude ester. The crude material was then dissolvedin ethanol (200 mL) and water (20 mL). LiOH (8.6 g) was added and theresulting mixture was heated to reflux (80° C.) for 3 hours. Thesolution was cooled and the solvent removed. 150 mL of H₂O was added andthe aqueous solution was acidified to a pH of approximately 2 withconcentrated HCl. The flask was cooled by placing it in a 4° C.refrigerator for 4 hours. A tan colored solid precipitated. Thismaterial was collected by vacuum filtration and dried on the high vacuumovernight to give 18 g of material (74% yield). This was furtherpurified by recrystallization from ethyl acetate/hexanes (about 95/5) togive 15 g of 8-(2-cyanophenoxy)octanoic acid. Melting point: 83–85° C.Karl Fisher: 1.1%. Molecular Formula (with H₂O): C₁₅H₁₉NO₃*0.1613H₂O.Combustion analysis (with H₂O included): % C: 68.19 (calc'd), 68.53(found); % H: 7.37 (calc'd), 7.33 (found); % N: 5.30 (calc'd), 5.34(found). ¹H NMR Analysis: (d₆-DMSO): δ 12.0, s, 1H (COOH); δ 7.71–7.60,m, 2H (aromatic CH's ortho and para to CN); δ 7.23, d, 1H, J=8.4 Hz(aromatic CH para to OR); δ 7.10, dt, 1H, J=0.7 and 6.7 Hz (aromatic CHortho to OR); δ 4.13, t, 2H, J=6.4 Hz (CH₂ α to O); δ 2.22, t, 2H, J=7.3Hz (CH₂ α to COOH); δ 1.73, m, 2H, (CH₂ α to O); δ 1.52–1.28, m, 8H(remaining aliphatic CH₂'s).

1f. Preparation of Compound 6

A 3-neck 300 mL round-bottomed flask equipped with a reflux condenser,magnetic stir bar and a nitrogen inlet was charged with 4 g (1equivalent) of 2-hydroxybenzonitrile, absolute ethanol 140 mL, and 12.54mL (1 equivalent) of sodium ethoxide. This mixture was stirred at 25° C.for 15 minutes. Ethyl 10-bromodecanoate (9.4 g, 1 equiv.) was then addeddropwise over 10 minutes. The resulting mixture was heated to reflux(75° C.) for 72 hours.

The reaction mixture was cooled and the solids filtered off. The solventwas removed on a rotary evaporator. The crude residue was dissolved inethyl acetate (300 mL) and washed with ½ saturated NaHCO₃ (2×100 mL),H₂O (1×100 mL) and brine (1×50 mL). The organic layer was dried to give10 g of the crude ester. The crude material was then dissolved inethanol (100 mL) and water (20 mL). LiOH (3.3 g) was added and theresulting mixture was heated to reflux (75° C.) for 3 hours. Thesolution was cooled and the solvent removed. 20 mL of H₂O was added andthe aqueous solution was acidified to a pH of about 3 with concentratedHCl. The solution was transferred to a 4° C. refrigerator to cool. A tancolored solid began to precipitate. This material was collected byvacuum filtration and dried on the high vacuum overnight to give thecrude acid. These solids were further purified by recrystallization fromethyl acetate/hexanes (95/5) to give 7.1 g of the product,10-(2-cyanophenoxy)decanoic acid (78% yield). Melting point: 82–84° C.Molecular Formula with water: C₁₇H₂₃NO₃*0.0532. Combustion analysis: %C: 70.33 (calc'd), 69.82 (found); % H: 8.02(calc'd), 7.89(found); % N:4.82 (calc'd), 4.82 (found).

1g: Preparation of Compound 7

A 3-neck 300 mL round-bottomed flask equipped with a reflux condenser,magnetic stir bar and an N₂ inlet was charged with 5 g (1 equivalent) of2-hydroxy-5-chlorobenzonitrile, absolute ethanol 125 mL, and 12.16 mL (1equivalent) of sodium ethoxide. This mixture was stirred at 25° C. for15 minutes. Ethyl 5-bromovalerate (5.2 mL, 1 equivalent) was then addeddropwise over 10 minutes. The resulting mixture was heated to reflux(75° C.) for 72 hours.

The reaction mixture was cooled and the solids filtered off. The solventwas removed on a rotary evaporator. The crude residue was dissolved inmethylene chloride (200 mL) and washed with saturated NaHCO₃ (2×75 mL),H₂O (1×100 mL) and brine (1×100 mL). The crude material was thendissolved in ethanol (120 mL) and water (10 mL). LiOH (4 g) was addedand the resulting mixture was heated to reflux (75° C.) for 1 hours thenstirred at ambient temperature overnight. The solvent was evaporated and75 mL of H₂O was added. The aqueous solution was acidified to a pH ofabout 3 with concentrated HCl and the flask cooled to 4° C. Tan coloredsolids precipitated. This material was collected by vacuum filtrationand dried on the high vacuum overnight to give the crude acid. Thesesolids were further purified by recrystallization from ethylacetate/hexanes (95/5) (three times) to give 2.88 g of the product,5-(4-chloro-2-cyanophenoxy)pentanoic acid (35% yield). Melting point:87–90° C. Molecular Formula: C₁₂H₁₂ClNO₃. Combustion analysis: % C:56.82(calc'd), 57.03(found); % H: 4.77(calc'd), 4.71(found); % N:5.52(calc'd), 5.45(found); Cl 13.98(calc'd), 13.93(found).

1h: Preparation of Compound 8

A 3-neck 300 mL round-bottomed flask equipped with a reflux condenser,magnetic stir bar and a nitrogen inlet was charged with 5 g (1equivalent) of 4-hydroxybenzonitrile, absolute ethanol 150 mL, and 15.7mL (1 equivalent) of sodium ethoxide. This mixture was stirred at 25° C.for 15 minutes. Ethyl 8-bromooctanoate (10.5 g, 1 equivalent) was thenadded dropwise over 10 minutes. The resulting mixture was heated toreflux (75° C.) for 72 hours.

The reaction mixture was cooled and the solids filtered off. The solventwas removed on a rotary evaporator. The crude residue was dissolved inmethylene chloride (200 mL) and washed with saturated NaHCO₃ (2×75 mL),H₂O (1×100 mL) and brine (1×100 mL). The crude material was thendissolved in ethanol (125 mL) and water (10 mL). LiOH (5 g) was addedand the resulting mixture was heated to reflux (75° C.) for 1 hour thenstirred at ambient temperature overnight. The solvent was evaporated and75 mL of H₂O was added. The aqueous solution was acidified to a pH ofabout 3 with concentrated HCl and the flask cooled to 4° C. An off-whitecolored solid precipitated. This material was collected by vacuumfiltration and dried on the high vacuum overnight to give the crudeacid. These solids were further purified by recrystallization from Ethylacetate/hexanes (95/5) and again with chloroform to give 4.5 g of theproduct, 8-(4-cyanophenoxy)octanoic acid (41% yield). Melting point:137–140° C. Molecular Formula: C₁₅H₁₉NO₃. Combustion analysis: % C:68.94 (calc'd), 68.57 (found); % H: 7.33(calc'd), 7.13(found); % N: 5.36(calc'd), 5.28 (found).

1i: Preparation of Compound 9

Potassium hydroxide (15.02 g, 268.4 mmol) was ground in a mortar untilpowdered, then added to a 125 mL Erlenmeyer flask containing 50 mL ofdimethylsulfoxide (DMSO), 8 g (6.71 mmol) of 2-hydroxybenzonitrile and12.59 g (7.38 mmol) of 4-(chloromethyl)benzoic acid. The reaction wasstirred at room temperature for six days. Distilled water (200 mL) wasadded to the brown reaction mixture, and the resulting solution wascooled to 4° C. Once cooled, the solution was acidified withconcentrated HCl. The resulting solid was collected by vacuum filtrationthrough a Buchner funnel. This material was purified by repeatedrecrystallizations from ethyl acetate to give 5.71 g of the product,4-(2-cyanophenoxymethyl)benzoic acid. Melting point: 199–203° C.Combustion analysis: % C: 71.14 (calc'd), 70.89 (found); % H: 4.38(calc'd), 4.35 (found); % N: 5.53 (calc'd), 5.25 (found); 1H NMRAnalysis: (d6-DMSO): δ 8.0, d, 2H; δ 7.8, d, 1H; δ 7.75, t, 1H; δ 7.65,d, 2H; δ 7.4, d, 1H; δ 7.2, t, 1H; δ 5.44, s, 2H.

EXAMPLE 2 EXAMPLE 2A Oral and Intacolonic Delivery of Heparin

Oral gavage (PO) and intracolonic (IC) dosing solutions containing adelivery agent compound and heparin sodium USP in 25% aqueous propyleneglycol were prepared. Either the sodium salt of the delivery agentcompound was used or the free acid was converted to the sodium salt withone equivalent of sodium hydroxide. Typically, the delivery agentcompound and heparin (about 166–182 IU/mg) were mixed by vortex as drypowders. This dry mixture was dissolved in 25% v/v aqueous propyleneglycol, vortexed and placed in a sonicator (about 37° C.). The pH wasadjusted to about 7 (6.5 to 8.5) with aqueous NaOH (2N). The dosingsolution was sonicated to produce a clear solution. The final volume wasadjusted to 3.0 mL. The final delivery agent compound dose, heparin doseand volume dose amounts are listed below in Table 2.

The typical dosing and sampling protocols were as follows. MaleSprague-Dawley rats weighing between 275–350 g were fasted for 24 hoursand were anesthetized with ketamine hydrochloride (88 mg/kg)intramuscularly immediately prior to dosing. A dosing group of five ratswas administered one of the dosing solutions. For oral gavage (PO)dosing, an 11 cm Rusch 8 French catheter was adapted to a 1 mL syringewith a pipette tip. The syringe was filled with dosing solution bydrawing the solution through the catheter, which was then wiped dry. Thecatheter was placed down the esophagus leaving 1 cm of tubing past therat's incisors. Solution was administered by pressing the syringeplunger. For intracolonic (IC) dosing, a 7.5 cm 8 fr Rusch catheter wasadapted to a 1 ml syringe with a pipette tip. The dosing catheter wasinserted into the colon through the anus until the tube was no longervisible. The dosing solution was expressed slowly into the colon.

Citrated blood samples were collected by cardiac puncture following theadministration of ketamine (88 mg/kg), typically at time—0.25, 0.5, 1.0and 1.5 hours. Heparin activity was determined by utilizing theactivated partial thromboplastin time (APTT) according to the method ofHenry, J. B., Clinical Diagnosis and Management by Laboratory Methods,Philadelphia, Pa., W. B. Saunders (1979). Previous studied indicatedbaseline values of about 20 sec. Results from the five rats in eachgroup were averaged for each time point. The maximum is reported belowin Table 2.

TABLE 2 Oral and Intracolonic Delivery of Heparin Method of VolumeCompound Heparin Mean Peak Com- Admini- Dose Dose Dose APTT (sec) ±pound stration (ml/kg) (mg/kg) (mg/kg) SD) 3 IC 200 25 1 16.23 ± 1.23  3Oral 300 100 1 203.59 ± 72.97  5 IC 50 25 1 80.22 ± 45.70 5 Oral 300 1001 176.25 ± 175.01

EXAMPLE 2B Oral Delivery of Recombinant Human Growth Hormone (rhGH)

Oral gavage (PO) dosing solutions of delivery agent compound and rhGH inphosphate buffer were prepared. A solution of the delivery agentcompound was made either with the sodium salt of the compound or byconverting the free acid to its sodium salt. Typically, a solution ofthe delivery agent compound was prepared in phosphate buffer andstirred, adding one equivalent of sodium hydroxide (1.0 N) when makingsodium salt. The final dosing solutions were prepared by mixing thedelivery agent compound with an rhGH stock solution (15 mg rhGH/ml) anddiluting to the desired volume (usually 3.0 ml). The delivery agentcompounds and rhGH dose amounts are listed below in Table 3.

The typical dosing and sampling protocols were as follows. MaleSprague-Dawley rats weighing between 200–250 g were fasted for 24 hoursand administered ketamine (44 mg/kg) and chlorpromazine (1.5 mg/kg) 15minutes prior to dosing. A dosing group of five rats was administeredone of the dosing solutions. For oral gavage (PO) dosing, an 11 cm Rusch8 French catheter was adapted to a 1 mL syringe with a-pipette tip. Thesyringe was filled with dosing solution by drawing the solution throughthe catheter, which was then wiped dry. The catheter was placed down theesophagus leaving 1 cm of tubing past the rat's incisors. Solution wasadministered by pressing the syringe plunger.

Blood samples were collected serially from the tail artery, typically attime=0, 15, 30, 45, 60 and 90 minutes for oral dosing. The five samplesfrom each time period were pooled. Serum rHGH concentrations werequantified by an rHGH immunoassay test kit (Kit #K1F4015 from GenzymeCorporation Inc., Cambridge, Mass.). Previous studies indicated baselinevalues of about zero.

The maximum concentration for each group is reported below in Table 3.

TABLE 3 Oral Delivery of rhGH in Rats Compound rhGH Volume Peak DoseDose Dose Serum [rhGH] Compound (mg/kg) (mg/kg) (ml/kg) (ng/ml) ± SD(SE) 1 200 3 1 0 3 200 3 1 57.51 ± 60.97 (26.97) 4 200 3 1 11.52 ± 12.234 200 3 1 73.13 ± 73.69 9 200 3 1 57.53 ± 45.27 (20.24)

EXAMPLE 2C Oral Delivery of Cromolyn

Dosing solutions containing a delivery agent compound and cromolyn,disodium salt (cromolyn) (from Sigma Chemicals of St. Louis, Mo.) wereprepared in deionized water. The free acid of the delivery agentcompound was converted to the sodium salt with one equivalent of sodiumhydroxide. This mixture was vortexed and placed in a sonicator (about37° C.). The pH was adjusted to about 7–7.5 with aqueous NaOH.Additional NaOH was added, if necessary, to achieve uniform solubility,and the pH re-adjusted. The mixture was vortexed to produce a uniformsolution, also using sonication and heat if necessary. The deliveryagent compound solution was mixed with cromolyn from a stock solution(175 mg cromolyn/ml in deionized water, pH adjusted, if necessary, withNaOH or HCl to about 7.0, stock solution stored frozen wrapped in foil,then thawed and heated to about 30° C. before using). The mixture wasvortexed to produce a uniform solution, also using sonication and heatif necessary. The pH was adjusted to about 7–7.5 with aqueous NaOH. Thesolution was then diluted with water to the desired volume (usually 2.0ml) and concentration and stored wrapped in foil before use. The finaldelivery agent compound and cromolyn doses, and the dose volumes arelisted below in Table 4.

The typical dosing and sampling protocols were as follows. MaleSprague-Dawley rats weighing between 200–250 g were fasted for 24 hoursand were anesthetized with ketamine (44 mg/kg) and chlorpromazine (1.5mg/kg) 15 minutes prior to dosing and again as needed to maintainanesthesia. A dosing group of five animals was administered one of thedosing solutions. An 11 cm Rusch 8 French catheter was adapted to a 1 mlsyringe with a pipette tip. The syringe was filled with dosing solutionby drawing the solution through the catheter, which was then wiped dry.The catheter was placed down the esophagus leaving 1 cm of tubing pastthe incisors. Solution was administered by pressing the syringe plunger.

Blood samples were collected via the tail artery, typically at 0.25,0.5, 1.0 and 1.5 hours after dosing. Serum cromolyn concentrations weremeasured by HPLC. Samples were prepared as follows: 100 μl serum wascombined with 100 μl 3N HCl and 300 μl ethyl acetate in an eppendorftube. The tube was vortexed for 10 minutes and then centrifuged for 10minutes at 10,000 rpm. 200 μl ethyl acetate layer was transferred to aneppendorf tube containing 67 μl 0.1 M phosphate buffer. The tube wasvortexed for 10 minutes and then centrifuged for 10 minutes at 10,000rpm. The phosphate buffer layer was then transferred to an HPLC vial andinjected into the HPLC (column=Keystone Exsil Amino 150×2 mm i.d., 5 μm,100 Å; mobile phase 35% buffer(68 mM KH₂PO₄ adjusted to pH 3.0 with 85%H₃PO₄)/65% acetonitrile; injection volume=10 μl; flow rate=0.30ml/minute; cromolyn retention time=5.5 minutes; absorbance detected at240 nm). Previous studies indicated baseline values of about zero.

Results from the animals in each group were averaged for each time pointand the highest of these averages (i.e., mean peak serum cromolynconcentration) is reported below in Table 4.

TABLE 4 Cromolyn - Oral Delivery Mean Peak Compound Cromolyn Volumeserum [cromolyn] Dose Dose Dose (μg/ml) ± Compound (mg/kg) (mg/kg)(ml/kg) SD (SE) 3 200 25 1 0.62 ± 0.29 (0.13) 4 200 25 1 0.82 ± 0.65(0.29) 5 200 25 1 0.46 ± 0.22 (0.10) 9 200 25 1 0.40 ± 0.21 (0.10)Insulin—Oral Delivery

Oral dosing (PO) compositions of delivery agent compound and human zincinsulin (minimum 26 IU/mg available from Calbiochem—Novabiochem Corp, LaJolla, Calif.) were prepared in deionized water. Typically, 500 mg ofdelivery agent compound was added to 1.5 ml of water. The free acid ofthe delivery agent compound was converted to the sodium salt by stirringthe resultant solution and adding one equivalent of sodium hydroxide.The solution was vortexed, then heated (about 37° C.) and sonicated. ThepH was adjusted to about 7 to 8.5 with NaOH or HCl. Additional NaOH wasadded, if necessary, to achieve uniform solubility, and the pHre-adjusted to about 7 to 8.5. Water was then added to bring the totalvolume to about 2.4 ml and vortexed. About 1.25 mg insulin from aninsulin stock solution (15 mg/ml made from 0.5409 g insulin and 18 mldeionized water, adjusting with HCl and NaOH to pH 8.15 and to obtain aclear solution using 40 ml concentrated HCl, 25 ml 10N NaOH and 50 ml 1NNaOH) was added to the solution and mixed by inverting. The solution maybe used in the dosing protocol immediately, or alternatively, thesolution may be placed into a 37° C. water bath for one hour prior todosing. The final delivery agent compound dose, insulin dose and dosevolume amounts are listed below in Table 5.

The typical dosing and sampling protocols were as follows. MaleSprague-Dawley rats weighing between about 200–250 g were fasted for 24hours and administered ketamine (44 mg/kg) and chlorpromazine (1.5mg/kg) 15 minutes prior to dosing and again as needed to maintainanesthesia. A dosing group of five animals was administered one of thedosing solutions. For oral dosing, an 11 cm Rusch 8 French catheter wasadapted to a 1 ml syringe with a pipette tip. The syringe was filledwith dosing solution by drawing the solution through the catheter, whichwas then wiped dry. The catheter was placed down the esophagus leaving 1cm of tubing past the incisors. The dosing solution was administered bypressing the syringe plunger.

Blood samples were collected serially from the tail artery, typically attime=15, 30, 60, 120 and 180 minutes. Serum insulin levels weredetermined with an Insulin ELISA Test Kit (Kit # DSL-10-1600 fromDiagnostic Systems Laboratories, Inc., Webster, Tex.), modifying thestandard protocol in order to optimize the sensitivity and linear rangeof the standard curve for the volumes and concentrations of the samplesused in the present protocol. Serum human insulin concentrations (μU/ml)were measured for each time point for each of the five animals in eachdosing group. The five values for each time point were averaged and theresults plotted as serum insulin concentration versus time. (Previousexperiments revealed no measurable levels of human insulin followingoral dosing with human insulin alone.) The maximum (peak) and the areaunder the curve (AUC) are reported below in Table 5.

TABLE 5 Insulin - Oral Delivery De- Delivery livery Agent Agent Com-Com- pound Insulin Volume pound Dose Dose Dose Mean Peak # (mg/kg)(mg/kg) (ml/kg) Serum Human Insulin 1 200 0.5 1.0 218.74 ± 361.02 (IU/ml± SD) 2 200 0.5 1.0 595.45 ± 1123.42 (IU/ml ± SD) 2 200 0.5 1.0  22.88 ±34.87 (μU/ml ± SD) 3 200 0.5 1.0  1.57 ± 3.44 (μU/ml ± SD) 3 200 0.5 1.0338.67 ± 456.61 (μU/ml ± SD) 3 200 0.5 1.0  0.23 ± .60 (μU/ml ± SD) 3200 0.5 1.0 267.53 ± 586.97 (μU/ml ± SD) 3 200 0.5 1.0  0.48 ± 1.18(μU/ml ± SD) 3 200 0.5 1.0  89.53 ± 60.14 (μU/ml ± SD) 3 200 0.5 1.0 5.70 ± 4.04 (μU/ml ± SD) 3 200 0.5 1.0  18.24 ± 21.24 (μU/ml ± SD) 3200 0.5 1.0  5.81 ± 6.96 (μU/ml ± SD) 3 200 0.5 1.0 222.74 ± 135.16(μU/ml ± SD) 3 200 0.5 1.0 101.75 ± 79.39 (μU/ml ± SD) 4 200 0.5 1.0  559 ± 410 (μU/ml ± SD) 5 100 3 0.5 695.13 ± 921.15 (μU/ml ± SD) 5 2000.5 0.5 669.40 ± 847.88 (μU/ml ± SD) 5 200 0.5 1.0 109.37 ± 119.44(μU/ml ± SD) 5 200 0.5 1.0 185.76 ± 94.24 (μU/ml ± SD) 5 200 0.5 1.0153.19 ± 114.61 (μU/ml ± SD) 5 200 0.5 1.0 323.76 ± 177.89 (μU/ml ± SD)5 200 0.5 1.0  53.44 ± 39.90 (μU/ml ± SD) 5 200 0.5 1.0  99.83 ± 76.37(μU/ml ± SD) 6 200 0.5 1.0  0.33 ± 0.69 (μU/ml ± SD) 6 200 0.5 1.0  1.99± 3.29 (μU/ml ± SD) 7 200 0.5 1.0  62.15 ± 56.42 (μU/ml ± SD) 7 200 0.51.0  91.22 ± 44.59 (μU/ml ± SD) 8 200 0.5 1.0  8.18 ± 5.01 (μU/ml ± SD)9 200 0.5 1.0 443.31 ± 632.53 (μU/ml ± SD)

The above mentioned patents, applications, test methods, andpublications are hereby incorporated by reference in their entirety.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed description. Allsuch obvious variations are within the fully intended scope of theappended claims.

1. A compound selected from the group consisting of compounds having theformula:

and salts thereof, wherein R¹ and R⁵ are independently H, —CN, —OH, orhalogen; R², and R⁴ are independently H, —CN, —OH, —OCH₃, or halogen, atleast one of R¹, R², R⁴ or R⁵ being —CN; and R³ is H, —OH, —OCH₃ orhalogen; and R⁶ is C₂–C₁₂ linear or branched alkylene, alkenylene,arylene, alkyl(arylene) or aryl(alkylene).
 2. A pharmaceuticalcomposition comprising: (A) a biologically active agent; and (B) atleast one compound selected from the group consisting of compoundshaving the formula:

and salts thereof, wherein R¹, R², R³, R⁴ and R⁵ are independently H,—CN, —OH, —OCH₃, or halogen, at least one of R¹, R², R³, R⁴ or R⁵ being—CN; and R⁶ is C₁–C₁₂ linear or branched alkylene, alkenylene, arylene,alkyl(arylene) or aryl(alkylene), with the proviso that when R¹ is —CN,R⁴ is H or —CN, and R², R³, and R⁵ are H, then R⁶ is not (CH₂)₁, whereinthe biologically active agent comprises at least one protein,polypeptide, peptide, hormone, polysaccharide, mucopolysaccharide,carbohydrate, or lipid.
 3. A pharmaceutical composition comprising: (A)a biologically active agent; and (B) at least one compound selected fromthe group consisting of compounds having the formula:

and salts thereof, wherein R¹, R², R³, R⁴ and R⁵ are independently H,—CN, —OH, —OCH₃, or halogen, at least one of R¹, R², R³, R⁴ or R⁵ being—CN; and R⁶ is C₁–C₁₂ linear or branched alkylene, alkenylene, arylene,alkyl(arylene) or aryl(alkylene), with the proviso that when R¹ is —CN,R⁴ H or —CN, and R², R³, and R⁵ are H, then R⁶ is not (CH₂)₁, whereinthe biologically active agent is selected from the group consisting of:growth hormones, human growth hormones (hGH), recombinant human growthhormones (rhGH), bovine growth hormones, porcine growth hormones, growthhormone-releasing hormones, interferons, α-interferon, β-interferon,γ-interferon, interleukin-1, interleukin-2, insulin, porcine insulin,bovine insulin, human insulin, human recombinant insulin, insulin-likegrowth factor (IGF), IGF-1, heparin, unfractionated heparin,heparinoids, dermatans, chondroitins, low molecular weight heparin, verylow molecular weight heparin, ultra low molecular weight heparin,unfractionated heparin, heparinoids, dermatans, chondroitins, lowmolecular weight heparin, very low molecular weight heparin, ultra lowmolecular weight heparin, calcitonin, salmon calcitonin humancalcitonin; erythropoietin (EPO), atrial naturetic factor, antigens,monoclonal antibodies, somatostatin, protease inhibitors,adrenocorticotropin, gonadotropin releasing hormone, oxytocin,leutinizing-hormone-releasing-hormone, follicle stimulating hormone,glucocerebrosidase, thrombopoietin, filgrastim, prostaglandins,cyclosporin, vasopressin, cromolyn sodium, sodium chromoglycate,disodium chromoglycate, vancomycin, desferrioxamine (DFO), parathyroidhormone (PTH), fragments of PTH, antimicrobials, antibiotics,antibacterials, anti-fungal agents, daptomycin, vitamins; mimetics andpolyethylene glycol (PEG)-modified derivatives of these compounds; andany combination thereof.
 4. The composition of claim 3, wherein thebiologically active agent comprises hGH, cromolyn sodium, sodiumchromoglycate, disodium chromoglycate, insulin, porcine insulin, bovineinsulin, human insulin, human recombinant insulin, insulin-like growthfactor (IGF), IGF-1, antimicrobials, antibiotics, antibacterial agents,anti-fungal agents, daptomycin, mimetics and polyethylene glycol(PEG)-modified derivatives of these compounds, or combinations thereof.5. The composition of claim 3, wherein the biologically active agentcomprises cromolyn sodium.
 6. The composition of claim 3, wherein thebiologically active agent comprises heparin.
 7. The composition of claim3, wherein the biologically active agent comprises insulin.
 8. Thecomposition of claim 3, wherein the biologically active agent compriseshuman growth hormone.
 9. A dosage unit form comprising: (A) thecomposition of claim 4; and (B) (a) an excipient (b) a diluent, (c) adisintegrant, (d) a lubricant, (e) a plasticizer, (f) a colorant, (g) adosing vehicle, or (h) any combination thereof.
 10. The dosage unit formof claim 9, wherein the biologically active agent is selected from thegroup consisting of: growth hormones, human growth hormones (hGH),recombinant human growth hormones (rhGH), bovine growth hormones,porcine growth hormones, growth hormone-releasing hormones, interferons,α-interferon, β-interferon, γ-interferon, interleukin-1, interleukin-2,insulin, porcine insulin, bovine insulin, human insulin, humanrecombinant insulin, insulin-like growth factor (IGF), IGF-1, heparin,unfractionated heparin, heparinoids, dermatans, chondroitins, lowmolecular weight heparin, very low molecular weight heparin, ultra lowmolecular weight heparin, calcitonin, salmon calcitonin humancalcitonin; erythropoietin (EPO), atrial naturetic factor, antigens,monoclonal antibodies, somatostatin, protease inhibitors,adrenocorticotropin, gonadotropin releasing hormone, oxytocin,leutinizing-hormone-releasing-hormone, follicle stimulating hormone,glucocerebrosidase, thrombopoietin, filgrastim, prostaglandins,cyclosporin, vasopressin, cromolyn sodium, sodium chromoglycate,disodium chromoglycate, vancomycin, desferrioxamine (DFO), parathyroidhormone (PTH), fragments of PTH, antimicrobials, antibiotics,antibacterials, anti-fungal agents, daptomycin, vitamins; mimetics andpolyethylene glycol (PEG)-modified derivatives of these compounds; andany combination thereof.
 11. The dosage unit form of claim 10, whereinthe biologically active agent comprises hGH, cromolyn sodium, sodiumchromoglycate, disodium chromoglycate, insulin, porcine insulin, bovineinsulin, human insulin, human recombinant insulin, insulin-like growthfactor (IGF), IGF-1, antimicrobials, antibiotics, antibacterial agents,anti-fungal agents, daptomycin, mimetics and polyethylene glycol(PEG)-modified derivatives of these compounds, or combinations thereof.12. The dosage unit form of claim 9, wherein the dosage unit form is inthe form of a tablet, a capsule, a particle, a powder, a sachet, or aliquid.
 13. The dosage unit form of claim 9, wherein the dosing vehicleis a liquid selected from the group consisting of water, 25% aqueouspropylene glycol, phosphate buffer, 1,2-propane diol, ethanol, and anycombination thereof.
 14. A method for administering a biologicallyactive agent to an animal in need of the agent, the method comprisingadministering orally to the animal a pharmaceutical compositioncomprising: (A) the biologically active agent; and (B) at least onecompound selected from the group consisting of compounds:

and salts thereof, wherein R¹, R², R³, R⁴ and R⁵ are independently H,—CN, —OH, —OCH₃, or halogen, at least one of R¹, R², R³, R⁴ or R⁵ being—CN; and R⁶ is C₁–C₁₂ linear or branched alkylene, alkenylene, arylene,alkyl(arylene) or aryl(alkylene), with the proviso that when R¹ is —CN,R⁴ is H or —CN, and R², R³, and R⁵ are H, then R⁶ is not (CH₂)₁.
 15. Amethod for preparing a pharmaceutical composition comprising mixing: (A)at least one biologically active agent, wherein the biologically activeagent comprises at least one protein, polypeptide, peptide, hormone,polysaccharide, mucopolysaccharide, carbohydrate, or lipid; (B) at leastone compound selected from compounds having the formula:

and salts thereof, wherein R¹, R², R³, R⁴ and R⁵ are independently H,—CN, —OH, —OCH₃, or halogen, at least one of R¹, R², R³, R⁴ or R⁵ being—CN; and R⁶ is C₁–C₁₂ linear or branched alkylene, alkenylene, arylene,alkyl(arylene) or aryl(alkylene), with the proviso that when R¹ is —CN,R⁴ is H or —CN, and R², R³, and R⁵ are H, then R⁶ is not (CH₂)₁; and (C)optionally, a dosing vehicle.
 16. A compound selected from the groupconsisting of

and salts thereof.
 17. A pharmaceutical composition comprising: (A) abiologically active agent; and (B) a delivery agent selected from thegroup consisting of

salts thereof, and mixtures thereof.
 18. The composition of claim 17,wherein the biologically active agent comprises at least one protein,polypeptide, peptide, hormone, polysaccharide, mucopolysaccharide,carbohydrate, or lipid.
 19. A method for preparing a pharmaceuticalcomposition comprising mixing: (A) at least one biologically activeagent, wherein the biologically active agent comprises at least oneprotein, polypeptide, peptide, hormone, polysaccharide,mucopolysaccharide, carbohydrate, or lipid; (B) a compound selected from

and salts thereof; and (C) optionally, a dosing vehicle.
 20. A compoundselected from the group consisting of compounds having the formula:

and salts thereof, wherein R¹, R², R⁴ and R⁵ are independently H, —CN,—OH, —OCH₃, or halogen, at least one of R¹, R², R⁴ or R⁵ being —CN; andR³ is H, —OH, —OCH₃, or halogen; and R⁶ is C₂–C₁₂ linear or branchedalkylene, alkenylene, arylene, alkyl(arylene) or aryl(alkylene).
 21. Thecompound of claim 20, wherein R⁶ is C₂–C₉ alkylene.
 22. The compound ofclaim 21, wherein R⁶ is C₄–C₇ alkylene.
 23. The compound of claim 21,wherein R⁶ is C₇ alkylene.
 24. A compound selected from the groupconsisting of compounds having the formula:

and salts thereof, wherein R¹, R², R⁴ and R⁵ are independently H, —CN,—OH, —OCH₃, or halogen, at least one of R¹ or R⁵ being —CN; and R³ is H,—OH, —OCH₃, or halogen; and R⁶ is C₂–C₁₂ linear or branched alkylene,alkenylene, arylene, alkyl(arylene) or aryl(alkylene).
 25. The compoundof claim 24, wherein R¹ is —CN.
 26. The compound of claim 25, wherein R⁶is C₂–C₉ alkylene.
 27. The compound of claim 25, wherein R⁶ is C₄–C₇alkylene.
 28. The compound of claim 24, wherein R⁶ is C₂–C₉ alkylene.29. The compound of claim 24, wherein R⁶ is C₄–C₇ alkylene.
 30. Thecompound of claim 1, wherein R⁶ is C₂–C₉ alkylene.
 31. The compound ofclaim 1, wherein R⁶ is C₄–C₇ alkylene.
 32. A pharmaceutical compositioncomprising: (A) a biologically active agent; and (B) at least onecompound selected from the group consisting of compounds having theformula:

and salts thereof, wherein R¹, R², R⁴, and R⁵ are independently H, —CN,—OH, —OCH₃, or halogen, at least one of R¹, R², R⁴, or R⁵ being —CN; andR³ is H, —OH, —OCH₃, or halogen; and R⁶ is C₁–C₁₂ linear or branchedalkylene, alkenylene, arylene, alkyl(arylene) or aryl(alkylene), withthe proviso that when R¹ is —CN, R⁴ is H or —CN, and R², R³, and R⁵ areH, then R⁶ is not (CH₂)₁.
 33. A dosage unit form comprising: (A) thecomposition of claim 32; and (B) (a) an excipient (b) a diluent, (c) adisintegrant, (d) a lubricant, (e) a plasticizer, (f) a colorant, (g) adosing vehicle, or (h) any combination thereof.
 34. The pharmaceuticalcomposition of claim 2, wherein the pharmaceutical composition is asolid pharmaceutical composition.
 35. The pharmaceutical composition ofclaim 17, wherein the pharmaceutical composition is a solidpharmaceutical composition.
 36. The pharmaceutical composition of claim32, wherein the pharmaceutical composition is a solid pharmaceuticalcomposition.
 37. The compound of claim 16, wherein the compound isselected from the group consisting of:

and salts thereof.
 38. The pharmaceutical composition of claim 2,wherein the compound is selected from

and salts thereof.